This invention relates to the application of recombinant DNA procedures to the production of useful enzymes. More specifically, it relates to the cloning and expression of a reverse transcriptase from a new source, Moloney murine leukemia virus, and to methods for the large-scale preparation of the enzyme so cloned.
The RNA tumor viruses cause the integration into host cell DNA of genetic material coding for the production of several proteins. Among these are the reverse transcriptase (hereinafter RT) enzymes, which direct the synthesis of complementary DNA from an RNA template. One member of this family of enzymes has been found to have great utility, and has been widely used.
Avian myeloblastosis virus (AMV) RT has been an essential tool in recombinant DNA technology. During the course of its use, numerous studies have been performed attempting to establish conditions optimal for the synthesis of full-length complementary DNA in high yield from mRNA (see Berger, S.L., et al., Reverse transcriptase and its associated ribonuclease H: interplay of two enyzmatic activities controls the yield of single-stranded complementary deoxyribonucleic acid. Biochemistry 22 (1983) 2365-2372.) Recent evidence indicates that nucleolytic activities that are an integral part of the enzyme (i.e., RNase H or DNA endonuclease; Gerard, G.F.: Reverse transcriptase in Jacob, S.T. (Ed.), Enzymes of Nucleic Acid Synthesis and Modification, Vol. I, DNA Enzymes. CRC Press, Boca Raton, FL, 1983, pp. 1-38.) or associated contaminants have strikingly adverse effects upon the length and yield of complementary DNA during synthesis (Berger et al., 1983).
Cloning and over-expression of the enzyme in E. coli should afford the opportunity to engineer and purify a form of RT more suitable for applications in recombinant DNA technology. However, there are apparent obstacles to expressing AMV RT in bacteria. In particular, to generate native AMV RT, the avian polymerase gene product must form a dimer that is phosphorylated and cleaved proteolytically at a specific site in one subunit, events that are not catalyzed in E. coli.
Accordingly, it is an object of the present invention to provide an improved form of reverse transcriptase, and genetic material coding therefor, which is suitable for use in recombinant DNA technology.
A further object of the invention is to provide improved means for the expression of the RT genetic material.
Yet another object of the invention is to provide improved means for the purification of the RT enzyme so expressed.